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PDBsum entry 1kbb

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protein metals links
Hydrolase PDB id
1kbb
Jmol
Contents
Protein chain
496 a.a. *
Metals
_CL
_CA
Waters ×120
* Residue conservation analysis
PDB id:
1kbb
Name: Hydrolase
Title: Mechanistic analyses of catalysis in human pancreatic alpha- amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids
Structure: Alpha-amylase, pancreatic. Chain: a. Synonym: 1,4-alpha-d-glucan glucanohydrolase, pancreatic alpha-amylase. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: pichia pastoris. Expression_system_taxid: 4922
Resolution:
1.90Å     R-factor:   0.172     R-free:   0.194
Authors: E.H.Rydberg,C.Li,R.Maurus,C.M.Overall,G.D.Brayer,S.G.Withers
Key ref:
E.H.Rydberg et al. (2002). Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids. Biochemistry, 41, 4492-4502. PubMed id: 11914097 DOI: 10.1021/bi011821z
Date:
05-Nov-01     Release date:   10-Apr-02    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P04746  (AMYP_HUMAN) -  Pancreatic alpha-amylase
Seq:
Struc:
511 a.a.
496 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.1  - Alpha-amylase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   3 terms 
  Biological process     metabolic process   5 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
DOI no: 10.1021/bi011821z Biochemistry 41:4492-4502 (2002)
PubMed id: 11914097  
 
 
Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.
E.H.Rydberg, C.Li, R.Maurus, C.M.Overall, G.D.Brayer, S.G.Withers.
 
  ABSTRACT  
 
The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
19788271 L.M.Willis, R.Zhang, A.Reid, S.G.Withers, and W.W.Wakarchuk (2009).
Mechanistic investigation of the endo-alpha-N-acetylgalactosaminidase from Streptococcus pneumoniae R6.
  Biochemistry, 48, 10334-10341.  
17687508 B.Cobucci-Ponzano, F.Conte, M.Rossi, and M.Moracci (2008).
The alpha-L: -fucosidase from Sulfolobus solfataricus.
  Extremophiles, 12, 61-68.  
18214874 C.A.Tarling, K.Woods, R.Zhang, H.C.Brastianos, G.D.Brayer, R.J.Andersen, and S.G.Withers (2008).
The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts.
  Chembiochem, 9, 433-438.  
18613721 S.Cheluvaraja, M.Mihailescu, and H.Meirovitch (2008).
Entropy and free energy of a mobile protein loop in explicit water.
  J Phys Chem B, 112, 9512-9522.  
17592362 R.Quezada-Calvillo, C.C.Robayo-Torres, Z.Ao, B.R.Hamaker, A.Quaroni, G.D.Brayer, E.E.Sterchi, S.S.Baker, and B.L.Nichols (2007).
Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.
  J Pediatr Gastroenterol Nutr, 45, 32-43.  
  16511271 S.Z.Fisher, L.Govindasamy, C.Tu, M.Agbandje-McKenna, D.N.Silverman, H.J.Rajaniemi, and R.McKenna (2006).
Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 88-93.  
15722449 R.Maurus, A.Begum, H.H.Kuo, A.Racaza, S.Numao, C.Andersen, J.W.Tams, J.Vind, C.M.Overall, S.G.Withers, and G.D.Brayer (2005).
Structural and mechanistic studies of chloride induced activation of human pancreatic alpha-amylase.
  Protein Sci, 14, 743-755.
PDB codes: 1xgz 1xh0 1xh1 1xh2
15182367 N.Ramasubbu, C.Ragunath, P.J.Mishra, L.M.Thomas, G.Gyémánt, and L.Kandra (2004).
Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.
  Eur J Biochem, 271, 2517-2529.
PDB codes: 1jxj 1nm9
15304511 S.Numao, I.Damager, C.Li, T.M.Wrodnigg, A.Begum, C.M.Overall, G.D.Brayer, and S.G.Withers (2004).
In situ extension as an approach for identifying novel alpha-amylase inhibitors.
  J Biol Chem, 279, 48282-48291.
PDB codes: 1u2y 1u30 1u33
12975375 C.A.Tarling, S.He, G.Sulzenbacher, C.Bignon, Y.Bourne, B.Henrissat, and S.G.Withers (2003).
Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis.
  J Biol Chem, 278, 47394-47399.  
12930989 J.E.Nielsen, and J.A.McCammon (2003).
Calculating pKa values in enzyme active sites.
  Protein Sci, 12, 1894-1901.  
12702721 Y.Tatara, B.R.Lee, T.Yoshida, K.Takahashi, and E.Ichishima (2003).
Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis.
  J Biol Chem, 278, 25289-25294.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.